DESCRIPTION (Adapted from application abstract): The long-term research efforts will eventually encompass two species of the Hantavirus genera, the Hantaan virus and the Sin Nombre Virus (SNV). The Hantaan virus is associated with hemorrhagic fever with renal syndrome (HFRS), while SNV is a newly-recognized species that is now implicated in the recent outbreak of adult respiratory distress syndrome. Unlike HFRS, the Hantavirus pulmonary syndrome (HPS), causes a lethal respiratory illness without renal disease or hemorrhage. While the targeted tissues and the illnesses produced by SNV and Hantaan infections are different, their genomic sequences, protein products, and presumably their life cycles are similar. Therefore, it is important to gain an insight into how this family of viruses cycles through the cell. The proposed research will initially focus on the Hantaan virus as this species is better understood. Strategic approaches will then be applied to study SNV. The overall research objective will be to establish and evaluate methods to study the genomic components that are required for transcription. The approach will be to use a sensitive reporter gene, bearing 5' and 3' flanking (noncoding) regions of the S segment in the minus sense orientation, which represents the same orientation of the 5' and 3' noncoding regions of the S segment. Production of the minus sense S-reporter RNA will be made from plasmids transfected into Vero E6 cells. Alternatively, S-reporter RNA will be directly transfected into cells. Cells will be infected with virus prior to or following transfection at different time points. Transcription of the reporter gene by the hantavirus replication machinery will result in a positive sense mRNA encoding an active reporter enzyme. Transcription will be measured by direct visualization or by enzyme assays, while replication of cRNA and viral (v)RNA will be monitored by reverse transcriptase-polymerase chain reaction (RT-PCR). Once a successful reporter system is generated, the vRNA's extracistronic 5' and 3' flanking regions required for transcription will be delineated. The research will provide information pertinent to rationale drug design. Further, studies on replication will increase our understanding of the ability of the hantavirus genome to adapt to environmental or man-made (drug) stress.